1. Field of the Invention
The present invention relates generally to immunological assays and methods to measure biological compounds. More specifically, the present invention relates to novel methods of measuring Anti-Müllerian Hormone (AMH) in a sample, including a mammalian sample such as a human, mouse or rat sample. In particular, antibodies are provided that bind to the mature region of Anti-Mullerian Hormone.
2. Description of the Related Art
Anti-Müllerian Hormone (AMH), also known as Müllerian-Inhibiting Substance (MIS), is a 140-kilodalton (kDa) dimeric glycoprotein hormone belonging to the transforming growth factor β (TGF β) superfamily, which includes TGF-β and the various inhibin and activin glycoproteins (Teixeira et al., 2001). All members of this superfamily are dimeric glycoproteins, and all are involved in the regulation of tissue growth and differentiation. In common with other TGF-β proteins, AMH is synthesized as a large precursor with a short signal sequence followed by the pre-pro hormone that forms homodimers. Prior to secretion, the mature hormone undergoes glycosylation and dimerisation to produce a 140-kDa dimer of identical disulphide-linked 70-kDa monomer subunits; each monomer contains an N-terminal domain (also called the “pro” region) and a C-terminal domain (also called the “mature” region). In contrast to other TGF-β superfamily members, it is believed that AMH requires the N-terminal domain to potentiate activity of the C-terminal domain to attain full bioactivity (Wilson et al., 1993). Between 5-20% of AMH is then cleaved at a specific site between the N-terminal domain (the pro region) and the C-terminal domain (the mature region) of the 70-kDa monomer during cytoplasmic transit, to form two polypeptides of 58 kDa (pro region) and 12 kDa (mature region). These two parts of the monomer remain in non-covalent attachment. The human gene coding for AMH has been sequenced and isolated, and is located on the short arm of chromosome 19 (Picard et al., 1986). The structure of a specific receptor for AMH has also been isolated and characterized (di Clemente et al., 1994; Barrends et al., 1995). Across species, AMH retains 11 to 12 conserved cystine residues, of which seven are located in the mature region. This region demonstrates the greatest degree of amino acid sequence homology between species, with 108 of the last 112 residues being conserved between the rat and human sequences (Lee et al., 1993).
AMH has an important role in sexual differentiation during development. AMH is produced by the Sertoli cells of the testis in the male, and by ovarian granulosa cells in the female. During fetal development in males, secretion of AMH from testicular Sertoli cells is essential for the regression of the Mullerian ducts, and thus the normal development of the male reproductive tract (Picon et al., 1969). The Mullerian ducts are the primordium for the uterus, Fallopian tubes, and upper part of the vagina in the female. In the male, secretion of AMH by the Sertoli cells commences during embryogenesis and continues throughout life. Levels drop following puberty, decreasing slowly to a relatively low post-puberty value (Teixeira et al., 2001). In the female, serum AMH is maintained at relatively low levels when compared to the male. After puberty, when menstrual cycling begins, circulating AMH slowly decreases throughout life and becomes undetectable at menopause. In mice, ablation of AMH function causes increased loss of ovarian follicles and premature cessation of ovarian cycling (Durlinger et al., 1999).
Several clinical applications for measuring serum AMH in humans have been identified. Among these are the diagnosis of intersex disorders in children (Lee et al., 2003), precocious puberty and the delayed onset of puberty, cryptorchidism, anorchidism, and evaluation of male gonadal function (Teixeira et al., 2001). Other potential applications include the investigation of the peri-menopausal transition in women, and the detection and monitoring of granulosa cell cancer patients (Long et al., 2000). Recent work has shown AMH to have potential as a circulating marker for assessment of ovarian reserve and fertility in women (van Rooij et al., 2002; te Velde et al., 2002; Gruijters et al., 2003). After many years in which AMH could be described as an esoteric analyte, there is now considerable interest in its potential as a routine clinical marker.
Almost all previous AMH research studies have been carried out with one of two immunoassays. One assay developed by Hudson et al. (Hudson et al., 1990) uses two monoclonal antibodies raised against human recombinant AMH, both being directed to epitopes in the pro region of the molecule. Use of this assay to measure AMH in humans from infancy to adulthood has been reported by Lee et al. (1996). Lee et al. (1996) also report that AMH is stable when stored at −20° C. for up to 2 years or for up to three freeze-thaw cycles, but that measured values increased by 2- to 3-fold beyond 2 years of storage, and decreased by 50% or more after three freeze-thaw cycles. In addition, Lee et al. (1996) report that the antibodies used in the Hudson assay do not recognize the carboxy-terminal fragment (mature region) of AMH, and while they are highly specific for full-length unprocessed human AMH (unprocessed meaning not being cleaved between the pro and mature regions of the 70-kDa monomer), they have a much lower affinity for the amino-terminal fragment (pro region) than for the unprocessed protein. Accordingly, some of the individual variability in AMH concentrations reported by Lee et al. (1996) may be due to differences in the extent of processing of the AMH protein that occurs in vivo. Lee et al. (1996) also report that the Hudson assay antibodies recognize non-human primate as well as human AMH, but that they do not recognize bovine or rodent AMH. A second assay employed in AMH research studies uses a pair of monoclonal antibodies, one of which is to an epitope in the pro region and the other is to an epitope in the mature region of human AMH (Long et al., 2000). A third more recently described assay uses two monoclonal antibodies against the pro region (Al-Qahtani et al., 2005).
Proteolysis of AMH in samples measured using currently available assays has also been reported, so that particularly careful attention to sample collection and storage may be required if reliable results are to be obtained. The pro region of AMH is subject to proteolytic cleavage during incubation in solution (Cate et al., U.S. Pat. No. 5,359,033). The mature region of AMH is more stable against proteolysis compared to the pro region, in part because of its multiple cystine residues.
Previous AMH assays measure human AMH but cannot be used to measure AMH in rodent samples, probably because the amino acid sequence of the pro region varies considerably between species. The overall amino acid sequence homology between the pro regions in mouse, rat, human, bovine and chicken AMH varies from 37-89% (GenBank, National Centers for Biotechnology Information (NCBI) genetic database). Assays that can be used to measure AMH in multiple species, including mammalian species such as the mouse and rat as well as humans, have not previously been available in the art. In addition to clinical applications, such assays would have useful applications in research related to various clinical and other applications of AMH measurements. In addition, applications for the measurement of AMH would benefit from assays providing stable and accurate measured values of AMH in samples, including AMH measurements that are unaffected by proteolysis of AMH.
The present invention provides compositions and methods to measure AMH in a sample, and antibodies that bind to the mature region of AMH. The present invention also provides compositions and methods to measure AMH in biological samples, including mammalian samples such as human, monkey, mouse, rat, bovine and horse samples. In addition, the present invention provides compositions and methods to measure AMH in a sample, wherein the amount of AMH measured is not affected by proteolysis of AMH in the sample. Thus, the present invention fulfills these longstanding needs and desires in the art.